Filter binding test

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A filter binding assay , also filter binding assay (engl. Filter binding assay ), or membrane binding assay is a biochemical detection method for the interaction of molecules , such as protein-DNA interactions . The filter binding test is a form of the ligand binding test .

principle

The assay is based on the fact that a substance that is retained on a filter membrane due to its affinity is filtered together with the ligands to be examined . This is followed by a washing process to remove weakly bound molecules and detection of the bound ligands.

application

The filter binding test is used to detect and screen the interaction of molecules , one of which is bound to a membrane filter, for example a nitrocellulose ( cellulose nitrate ) filter . Among other things, the method is used to discover, map or characterize protein-DNA interactions . In this case, the assay relies on the fact that most proteins adhere to membrane filters while permeating double-stranded DNA. Developed in Paul Berg's laboratory to study RNA polymerase , the method was of great importance in understanding the regulation of the lac operon by the lac repressor . Although the method is older than any other evidence of protein-DNA interaction, it is still in use. The main reasons are the ease of implementation and the possibilities for automation. Among other things, it made it possible to demonstrate the specific binding of the Gal repressor to the operator of the Gal operon and of the TGGCA protein, which had not yet been purified, to the enhancer of the lysozyme gene . A disadvantage of the method is the inadequately understood interaction of proteins with the membrane (unspecific interaction) and the fact that proteins are sometimes inadequately retained.

method

The protein solution, either a cell extract , an enriched or a pure protein, is pipetted in a suitable buffer with the DNA to be examined onto a filter which rests on a filter apparatus connected to a vacuum pump . The solution is suctioned off and the filter is washed with the buffer. The DNA, usually a fragment or mixture of fragments produced by restriction digestion , or a synthetic DNA was previously radioactively labeled . The radioactivity can be determined as a direct measure of the bound DNA in a scintillation counter . The specificity of the binding can be proven by adding an excess of the unlabeled DNA compared to a non-specific DNA as a negative control , e.g. B. Cot-1 DNA . The investigation of fragment mixtures is followed by gel electrophoresis with subsequent autoradiography . The method is based on the work of Arthur Riggs and Suzanne Bourgeois at the Salk Institute . Alternative methods are e.g. B. EMSA and DNase Footprinting Assay .

Individual evidence

  1. ↑ R. Helwa, JD Hoheisel: Analysis of DNA-protein interactions: from nitrocellulose filter binding assays to microarray studies. In: Analytical Bioanalytical Chemistry Vol. 398, No. 6, 2010, pp. 2551-2561, PMID 20730525 .
  2. ↑ PG Stockley: Filter-binding assays. In: Methods Mol. Biol. Vol. 543, 2009, pp. 1-14, PMID 19378155
  3. ^ OW Jones, P. Berg Studies on the binding of RNA polymerase to polynucleotides. In: Journal of Molecular Biology Volume 22, Number 2, 1966, 199-209, doi : 10.1016 / 0022-2836 (66) 90126-4 , PMID 5972769
  4. ^ AD Riggs, S. Bourgeois, RF Newby, M. Cohn DNA binding of the lac repressor. In: Journal of Molecular Biology Volume 34, Number 6, 1968, 365-368, doi : 10.1016 / 0022-2836 (68) 90261-1 , PMID 4938552
  5. ↑ JS Park, M. Gottesman, K. Shimada, RA Weisberg, RL Perlman, I. Pastan: Isolation of the gal repressor In: Proceedings of the National Academy of Sciences . Vol. 68, No. 8, 1971, pp. 1891-1895, PMID 4942917 , / PMC 389315 (free full text).
  6. ↑ J. Nowock, AE Sippel: Specific protein-DNA interaction at four sites flanking the chicken lysozyme gene In: Cell Vol. 30, No. 2, 1982, pp. 607-615, PMID 6291778
  7. ↑ S. Oehler, R. Alex and A. Barker: Is nitrocellulose filter binding really a universal assay for protein-DNA interactions? In: Analytic Biochemistry Vol. 268, No. 2, 1999, pp. 330-336, PMID 10075823

literature